The primary objective of the proposed research is to identify the cellular mechanisms by which the heart responds to inotropic agents like catecholamines that produce their characteristic effects by activating myocardial adenylate cyclase. To attain this objective we propose to: 1. Reconstitute a calcium transport pump from solubilized cardiac sarcoplasmic reticulum (SR) with properties similar to those found in isolated SR. Reconstitution of a cardiac SR calcium pump will provide insight into its structure and function and will serve as a model for the SR of the intact myocardium. 2. Determine the mechanism of the regulation of the cardiac SR calcium pump by protein kinase-catalyzed phosphorylation of a 22,000 dalton protein, using the reconstituted membrane preparations developed in (1). Our previous studies have provided evidence for the following sequence of events: catecholamine binding to Beta-adrenergic receptors yields increased cyclic AMP production yields activation of cyclic AMP-dependent protein kinase yields phosphorylation of a 22,000 dalton protein of the cardiac SR yields increased rate of SR calcium transport yields increased rate of relaxation yields abbreviation of systole. We now propose to define the precise mechanism of the interaction of the 22,000 dalton protein with the cardiac SR calcium pump. 3. Initiate studies to elucidate the biochemical mechanism by which cyclic AMP mediates effects of catecholamines on cardiac sarcolemma. Specifically, search for protein substrates of cyclic AMP-dependent protein kinase, and if found, relate phosphorylation to sarcolemmal membrane function.